Abstract:

Zhang Liangyu, Wang Xiang, Lu Yaqi, Zhu Xiaoyang, Chen Yang. Department of Dermatology, 98th Hospital of PLA; 98 Clinical Medical College Affiliated to Anhui Medical University, Huzhou 313000, Zhejiang, China Corresponding author: Chen Yang, Email: 98cy@163.com 【Abstract】 Objective To estimate the effects of camptothecin （CPT） on the expression of hypoxia-inducible factor-1α （HIF-1α） in HaCaT cells under hypoxic conditions （2% O2）, and to explore the potential therapeutic mechanism of topical CPT for psoriasis. Methods Some HaCaT cells were classified into 6 groups: 5 test groups cultured in Dulbecco′s modified Eagle′s medium （DMEM） with the presence of CPT at 12.5, 25, 50, 100 and 200 nmol/L respectively, and 1 control group cultured in DMEM with the presence of dimethyl sulfoxide （DMSO）. All the 6 groups of cells were cultured under normoxic conditions for 12, 24, 48 or 72 hours or under hypoxic conditions for 12 hours. Cell counting kit-8 （CCK-8） assay was conducted to estimate the proliferation of HaCaT cells after the normoxic culture, and Western blot to quantify the protein expression of HIF-1α after the hypoxic culture. Some HaCaT cells were classified into a normoxia group （21% O2） and a hypoxia group （2% O2）, and each group was divided into a CPT （100 nmol/L）-treated subgroup and a non-intervention subgroup （treated with the vehicle）. After 12-hour culture, real-time fluorescence-based quantitative PCR was performed to measure the mRNA expression of HIF-1α. Statistical analysis was carried out by using Levene′.s test, one-way analysis of variance, Dunnett-t test and factorial analysis with the SPSS16.0 software. Results After treatment with CPT at 12.5 － 200 nmol/L for 12 － 72 hours, the proliferation of HaCaT cells was inhibited in a concentration- and time-dependent manner. More concretely, the cell proliferation rates were inhibited by 17.66% ± 6.46%, 33.11% ± 4.63% and 56.31% ± 1.69% respectively in HaCaT cells after 12-hour treatment with 200 nmol/L CPT as well as 24-hour treatment with 100 and 200 nmol/L CPT compared with the control group at the corresponding time points （all P < 0.05）. The protein expression level of HIF-1α was significantly decreased in HaCaT cells after 12-hour treatment with CPT at 12.5, 25, 50, 100 and 200 nmol/L under hypoxic conditions compared with the control group （0.348 ± 0.065, 0.261 ± 0.112, 0.115 ± 0.043, 0.045 ± 0.024 vs. 1.445 ± 0.329, all P < 0.05）. The mRNA expression level of HIF-1α （expressed as △Ct） in the CPT-treated subgroup and non-intervention subgroup was －5.575 ± 0.29 and －5.451 ± 0.21 respectively in the normoxia group, significantly higher than that in the hypoxia group （－6.543 ± 0.57 and －6.203 ± 0.31 respectively, F = 29.856, P < 0.05）, while there was no significant difference between the CPT-treated and non-intervention subgroups （F = 1.667, P > 0.05）. Conclusions CPT at 100 nmol/L could inhibit the expression of HIF-1α protein, but had no obvious effect on that of HIF-1α mRNA.